Further studies on the biosynthesis of mangiferin in Anemarrhena asphodeloides: hydroxylation of the shikimate-derived ring

The hydroxylation at C-3′ of maclurin, an intermediate in mangiferin biosynthesis, has been studied. Labelled cinnamic acid, p-coumaric acid, caffeic acid, iriflophenone and maclurin were fed to Anemarrhena asphodeloides. Cinnamic acid and p-coumaric acid were better precursors than caffeic acid for mangiferin, and iriflophenone as well as maclurin was effectively incorporated into mangiferin and isomangiferin. These results show that maclurin is biosynthesized via hydroxylation of iriflophenone derived from p-coumarate in this plant.     

UVB induced oxidative stress in human keratinocytes and protective effect of antioxidant agents

This study aims at exploring the oxidative stress in keratinocytes induced by UVB irradiation and the protective effect of nutritional antioxidants. Cultured Colo-16 cells were exposed to UVB in vitro followed by measurement of reactive oxygen species (ROS), endogenous antioxidant enzyme activity, as well as cell death in the presence or absence of supplementation with vitamin C, vitamin E, or Ginsenoside Panoxatriol. Intracellular ROS content was found significantly reduced 1 h after exposure, but increased at later time points. After exposure to 150–600 J m⁻² UVB, reduction of ROS content was accompanied by increased activity of catalase and CuZn-superoxide dismutase at early time points. Vitamins C and E, and Ginsenoside Panoxatriol counteracted the increase of ROS in the Colo-16 cells induced by acute UVB irradiation. At the same time, Ginsenoside Panoxatriol protected the activity of CuZn-superoxide dismutase, while vitamin E showed only a moderate protective role. Vitamins C and E, and Ginsenoside Panoxatriol in combination protected the Colo-16 cells from UVB-induced apoptosis, but not necrosis. These findings suggest that vitamins C and E as well as Ginsenoside Panoxatriol are promising protective agents against UVB-induced damage in skin cells.     

Circulating beta-chemokines and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 system in hemodialyzed patients--role of oxidative stress

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), beta-chemokines, increased oxidative stress (SOX) and inflammation have been implicated as important factors in atherosclerosis and vascular remodeling. We hypothesized the possible roles of beta-chemokines [monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins (MIP-1alpha, MIP-1beta) and regulated upon activation, normal T-cell expressed and secreted (RANTES)] as regulators of the metabolism of the vascular extracellular matrix in conditions of increased SOX in hemodialysis (HD) patients. We compared pre-dialysis levels of MMP-9/TIMP-1 system, beta-chemokines, Cu/Zn superoxide dismutase (Cu/Zn SOD) as a marker of SOX and C-reactive protein (CRP) as a marker of inflammation in HD patients with and without cardiovascular disease (CVD) to those of controls. HD patients, particularly those with CVD, showed a significant increase in values of Cu/Zn SOD, CRP, TIMP-1, TIMP-1/MMP-9 ratio, MCP-1 and MIP-1beta, whereas RANTES levels were lower than in the controls. The levels of MIP-1alpha as well as MMP-9 in all HD groups were similar to the controls. The positive correlations were observed between the MMP-9/TIMP-1 system and beta-chemokines, SOX and inflammation in whole HD group and in the subgroup with CVD. Multivariate analysis showed that the duration of dialysis followed by Cu/Zn SOD, MIP-1alpha and beta levels were the significant positive predictors of TIMP-1. In conclusion, our data show that MMP-9/TIMP-1 system and beta-chemokines could cooperate in conditions of elevated SOX, which ultimately predisposes hemodialysis patients to accelerated atherosclerosis.     

The apoptotic effect of sarsasapogenin from Anemarrhena asphodeloides on HepG2 human hepatoma cells

Sarsasapogenin, a kind of mainly effective components of Anemarrhena asphodeloides Bunge (Liliaceae) has the effects of being anti-diabetes and improving memory. However, there are few reports focusing on its anti-tumor effects. In this study, the sarsasapogenin was extracted from rhizomes of A. asphodeloides Bunge and applied to inhibit HepG2 human hepatoma cells. MTT assay showed that sarsasapogenin induced a distinct dose- and time-dependent diminution of cell viability with IC(50) of 42.4+/-1.0microg/ml for 48h. Furthermore, sarsasapogenin-induced apoptosis of HepG2 cells was verified by Hoechst 33258 staining, electron microscopy, DNA fragmentation and PI staining. Flow cytometry analysis showed that sarsasapogenin-induced cell apoptosis was through arrest of cell cycle in G(2)/M phase. Hence we proposed that sarsasapogenin could be used as an anti-liver cancer drug for future studies.     

Inhibition of matrix metalloproteinase-2 by PARP inhibitors

Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC₅₀ values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.     

Inhibitory effect of Aucubin isolated from Eucommia ulmoides against UVB-induced matrix metalloproteinase-1 production in human skin fibroblasts

Of 30 herbal plants tested, the methanol extracts of Eucommia ulmoides (52%), Evodia officinalis (45%), and Pleuropterus multiflorus (41%) each showed a potent inhibitory effect on the matrix metalloproteinase-1 (MMP-1) production in ultraviolet B (UVB)-irradiated human fibroblasts. Aucubin was isolated as the MMP-1 inhibitor from E. ulmoides, and significantly suppressed the production of MMP-1 by nearly 57% compared to the control. It also reduced MMP-1 mRNA expression. These results suggest that aucubin is a photoprotective phytochemical, and could be used as a potential agent in preventing photoaging.     

Prevention of selenite induced oxidative stress and cataractogenesis by luteolin isolated from Vitex negundo

Free radical mediated oxidative stress plays a crucial role in the pathogenesis of cataract and the present study was to determine the efficacy of luteolin in preventing selenite induced oxidative stress and cataractogenesis in vitro. Luteolin is a bioactive flavonoid, isolated and characterized from the leaves of Vitex negundo. Lenses were extracted from Sprague-Dawley strain rats and were organ cultured in DMEM medium. They were divided into three groups with eight lenses in each group as follows: lenses cultured in normal medium (G I), supplemented with 0.1mM sodium selenite (G II) and sodium selenite and 2 μg/ml luteolin (G III). Treatment was from the second to fifth day, while selenite administration was done on the third day. After the experimental period, lenses were taken out and various parameters were studied. The antioxidant potential of luteolin was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. In the selenite induced group, morphological examination of the lenses showed dense cortical opacification and vacuolization. Biochemical examinations revealed a significant decrease in activities of antioxidant enzymes and enzymes of the glutathione system. Additionally decreased glutathione level and increased reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were observed. Luteolin treatment abated selenite induced oxidative stress and cataractogenesis by maintaining antioxidant status, reducing ROS generation and lipid peroxidation in the lens. These finding demonstrated the anticataractogenic effect of luteolin by virtue of its antioxidant property, which has been reported in this paper for the first time.     

Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Americanin B protects cultured human keratinocytes against oxidative stress by exerting antioxidant effects

We evaluated the cytoprotective effects of americanin B, a lignan compound, against hydrogen peroxide (H₂O₂)-induced cell damage. Americanin B decreased the level of DPPH radicals, superoxide anions, hydroxyl radicals, and intracellular reactive oxygen species. Americanin B also attenuated DNA damage induced by H₂O₂ treatment, as shown by the inhibition of formation of comet tails, indicative of DNA strand breakage, and prevented the oxidation of protein and peroxidation of lipid, as determined by protein carbonyls and 8-isoprostane. Furthermore, americanin B protected against H₂O₂-induced apoptotic cell death, as determined by a reduction in the numbers of apoptotic bodies stained with Hoechst 33342. These findings suggest that americanin B protects cells against oxidative damage by exerting antioxidant effects and inhibiting apoptosis.     

Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes

BACKGROUND Matrix metalloproteinases(MMPs),including MMP-9,are an integral part of the immune response and are upregulated in response to a variety of stimuli.New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion.There is significant evidence for regulation of inflammation by dimethyl sulfoxide(DMSO)and 3',5'-cyclic adenosine monophosphate(cAMP),thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factorα(TNFα)secretion by human monocytes was of high interest.The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFαsecretion by distinct mechanisms.AIM To investigate the regulation of lipopolysaccharide(LPS)-stimulated MMP-9 and tumor necrosis factorαsecretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.METHODS The paper describes a basic research study using THP-1 human monocyte cells.All experiments were conducted at the University of Missouri-St.Louis in the Department of Chemistry and Biochemistry.Human monocyte cells were grown,cultured,and prepared for experiments in the University of Missouri-St.Louis Cell Culture Facility as per accepted guidelines.Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFαproduction.Inhibitors including DMSO,cAMP regulators,and anti-TNFαantibody were added to the cells prior to LPS treatment.MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software.TNFαsecretion was analyzed by enzyme-linked immuno sorbent assay.All data is presented as the average and standard error for at least 3 trials.Statistical analysis was done using a two-tailed paired Student t-test.P values less than 0.05 were considered significant and designated as such in the Figures.LPS and cAMP regulators were from Sigma-Aldrich,MMP-9 standard and antibody and TNFαantibodies were from R&D Systems,and amyloid-βpeptide was from rPeptide.RESULTS In our investigation of MMP-9 secretion from THP-1 human monocytes,we made the following findings.Inclusion of DMSO in the cell treatment inhibited LPSinduced MMP-9,but not TNFα,secretion.Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dosedependent fashion.A cell-permeable cAMP analog,dibutyryl cAMP,inhibited both LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFαin a dose-dependent fashion.Pre-treatment of monocytes with an anti-TNFαantibody blocked LPSinduced MMP-9 and TNFαsecretion.Amyloid-βpeptide induced MMP-9 secretion,which occurred much later than TNFαsecretion.The latter two findings strongly suggested an upstream role for TNFαin mediating LPS-stimulate MMP-9 secretion.CONCLUSION The cumulative data indicated that MMP-9 secretion was a distinct process from TNFαsecretion and occurred downstream.First,DMSO inhibited MMP-9,but not TNFα,suggesting that the MMP-9 secretion process was selectively altered.Second,cAMP inhibited both MMP-9 and TNFαwith a similar potency,but at different monocyte cell exposure time points.The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFαand that TNFαmay a key component of the pathway leading to MMP-9 secretion.This temporal relationship fit a model whereby early TNFαsecretion directly led to later MMP-9 secretion.Lastly,antibody-blocking of TNFαdiminished MMP-9 secretion,suggesting a direct link between TNFαsecretion and MMP-9 secretion.     

Exosomes derived from chemically induced human hepatic progenitors inhibit oxidative stress induced cell death

The emerging field of regenerative medicine has revealed that the exosome contributes to many aspects of development and disease through intercellular communication between donor and recipient cells. However, the biological functions of exosomes secreted from cells have remained largely unexplored. Here, we report that the human hepatic progenitor cells (CdHs)-derived exosome (EXO^hCdHs) plays a crucial role in maintaining cell viability. The inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease of cell viability. This event provokes inhibition of caspase dependent cell death signaling, leading to a ROS-dependent cell damage response and thus induces promotion of antioxidant gene expression or repair of cell death of hypoxia-exposed cells. Together, these findings show the effect of exosomes in regeneration of liver cells, and offer valuable new insights into liver regeneration.     

Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.     

UV-A induces persistent genomic instability in human keratinocytes through an oxidative stress mechanism

Ultraviolet-A (UV-A, 320 to 400 nm) radiation comprises 95% of the solar ultraviolet radiation (UVR) reaching the earth's surface. It has been associated experimentally and epidemiologically with malignant melanoma. In this study we investigated whether UV-A radiation can induce a persistent, heritable hypermutability in mammalian cells similar to that observed following ionising radiation (IR). Using the immortalized human skin keratinocyte cell line HaCaT we found that UV-A radiation does lead to a continuing reduction in plating efficiency, an increased "spontaneous" mutant fraction, and an increase in micronucleus formation up to 21 d after initial exposure. Reversal of these effects using catalase may indicate a role for hydrogen peroxide in this phenomenon. These results add to the significance of UV-A radiation as a risk factor in skin carcinogenesis.     

ER signaling is activated to protect human HaCaT keratinocytes from ER stress induced by environmental doses of UVB

Proteins are folded properly in the endoplasmic reticulum (ER). Various stress such as hypoxia, ischemia and starvation interfere with the ER function, causing ER stress, which is defined by the accumulation of unfolded protein (UP) in the ER. ER stress is prevented by the UP response (UPR) and ER-associated degradation (ERAD). These signaling pathways are activated by three major ER molecules, ATF6, IRE-1 and PERK. Using HaCaT cells, we investigated ER signaling in human keratinocytes irradiated by environmental doses of ultraviolet B (UVB). The expression of Ero1-Lα, an upstream signaling molecule of ER stress, decreased at 1-4 h after 10 mJ/cm² irradiation, indicating that the environmental dose of UVB-induced ER stress in HaCaT cells, without growth retardation. Furthermore, expression of intact ATF6 was decreased and it was translocated to the nuclei. The expression of XBP-1, a downstream molecule of IRE-1, which is an ER chaperone whose expression is regulated by XBP-1, and UP ubiquitination were induced by 10 mJ/cm² UVB at 4 h. PERK, which regulates apoptosis, was not phosphorylated. Our results demonstrate that UVB irradiation generates UP in HaCaT cells and that the UPR and ERAD systems are activated to protect cells from UVB-induced ER stress. This is the first report to show ER signaling in UVB-irradiated keratinocytes.